The overall objective of this project is to define how macrophage-produced type I IFN regulates activation for tumor cell killing. Our central hypothesis is that this autocrine/paracrine regulatory mechanism is important, both in the direct priming of macrophages and indirectly, as an agent that can affect the activation process by modulating the response of macrophages to other activating stimuli. We propose to test our hypothesis and to accomplish our overall objective by pursuing the following three specific aims. First, we will extensively characterize the biology of type I IFNs as they relate to activation for tumor cell killing in vitro and in vivo. We will begin by ascertaining whether different kinds of macrophages under different conditions of activation produce different spectra of type I IFNs. Once the relevant IFNs are known, their effects on the generation by macrophages of cytolytic mechanisms (TNF-alpha, H2O2, nitric oxide and IL-1) and other mediators that affect activation (PGE2 and TGF-beta) will be determined. Once these effects of macrophage-produced type I IFN have been defined, their role in augmenting/suppressing activation mediated by LPS alone and LPS in conjunction with combinations of type I and II IFNs will be determined. In separate experiments with peritoneal macrophages, we will determine whether or not endogenously-produced type I IFN is responsible for the reduced responsiveness to activating stimuli of resident compared to inflammatory peritonial macrophages. The second aim is to identify the mechanisms that lower the threshold of responsiveness to LPS and increase the production of type I IFN after macrophages are stimulated with IFN-gamma. We expect that eh decreased need for LPS will be due to synergism between IFN-gamma and type I IFN that IFN-gamma-primed macrophages produce in response to low concentrations of LPS. Alternative mechanisms to be investigated in collaboration with other project leaders include changes in other activation-related functional and biochemical pathways. Increased rate/duration of transcription, as well as changes in the stability of mRNA encoding macrophage interferons, will be sought as an explanation for increased production of type I IFN by IFN-gamma-primed macrophages. Finally, in aim #3, we will determine the mechanism by which pretreatment of macrophages with type I IFN suppresses their subsequent activation by IFN-gamma and LPS. There are several possibilities which might explain this phenomenon, e.g., down-regulation of receptors or alteration of biochemical pathways that are essential for activation. Many of these investigations will be conducted in collaboration with Drs. Morrison, Suzuki and Parmely. The studies proposed here are important because results will enhance overall understanding of how the process of macrophage activation is regulated by autocrine/paracrine feedback of type I IFNs.